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Boucher, J. M., Hanosset, R., Augot, D., Bart, J. M., Morand, M., Piarroux, R., et al. (2005). Detection of Echinococcus multilocularis in wild boars in France using PCR techniques against larval form. Vet Parasitol, 129(3-4), 259–266.
Abstract: Recently, new data have been collected on the distribution and ecology of Echinococcus multilocularis in European countries. Different ungulates species such as pig, goat, sheep, cattle and horse are known to host incomplete development of larval E. multilocularis. We report a case of E. multilocularis portage in two wild boars from a high endemic area in France (Department of Jura). Histological examination was performed and the DNA was isolated from hepatic lesions then amplified by using three PCR methods in two distinct institutes. Molecular characterisation of PCR products revealed 99% nucleotide sequence homology with the specific sequence of the U1 sn RNA gene of E. multilocularis, 99 and 99.9% nucleotide sequence homology with the specific sequence of the cytochrome oxydase gene of Echinococcus genus and 99.9% nucleotide sequence homology with a genomic DNA sequence of Echinococcus genus for the first and the second wild boar, respectively.
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Chilton, N. B. (2004). The use of nuclear ribosomal DNA markers for the identification of bursate nematodes (order Strongylida) and for the diagnosis of infections. Anim Health Res Rev, 5(2), 173–187.
Abstract: Many bursate nematodes are of major importance to animal health. Animals are often parasitized by multiple species that differ in their prevalence, relative abundance and/or pathogenicity. Implementation of effective management strategies for these parasites requires reliable methods for their detection in hosts, identification to the species level and measurement of intensity of infection. One major problem is the difficulty of accurately identifying and distinguishing many species of bursate nematode because of the remarkable morphological similarity of their eggs and larvae. The inability to identify, with confidence, individual nematodes (irrespective of their life-cycle stage) to the species level by morphological methods has often led to a search for species-specific genetic markers. Studies over the past 15 years have shown that sequences of the internal transcribed spacers of ribosomal DNA provide useful genetic markers, providing the basis for the development of PCR-based diagnostic tools. Such molecular methods represent powerful tools for studying the systematics, epidemiology and ecology of bursate nematodes and, importantly, for the specific diagnosis of infections in animals and humans, thus contributing to improved control and prevention strategies for these parasites.
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Gasser, R. B., Hung, G. - C., Chilton, N. B., & Beveridge, I. (2004). Advances in developing molecular-diagnostic tools for strongyloid nematodes of equids: fundamental and applied implications. Mol Cell Probes, 18(1), 3–16.
Abstract: Infections of equids with parasitic nematodes of the order Strongylida (subfamilies Strongylinae and Cyathostominae) are of major veterinary importance. In last decades, the widespread use of drugs against these parasites has led to problems of resistance within the Cyathostominae, and to an increase in their prevalence and intensity of infection. Novel control strategies, based on improved knowledge of parasite biology and epidemiology, have thus become important. However, there are substantial limitations in the understanding of fundamental biological and systematic aspects of these parasites, which have been due largely to limitations in their specific identification and diagnosis using traditional, morphological approaches. Recently, there has been progress in the development of DNA-based approaches for the specific identification of strongyloids of equids for systematic studies and disease diagnosis. The present article briefly reviews information on the classification, biology, pathogenesis, epidemiology of equine strongyloids and the diagnosis of infections, highlights knowledge gaps in these areas, describes recent advances in the use of molecular techniques for the genetic characterisation, specific identification and differentiation of strongyloids of equids as a basis for fundamental investigations of the systematics, population biology and ecology.
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Traversa, D., Giangaspero, A., Iorio, R., Otranto, D., Paoletti, B., & Gasser, R. B. (2004). Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces. Parasitology, 129(Pt 6), 733–739.
Abstract: Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.
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